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Polyphosphoinositides (PPIns) are a family of seven lipid messengers that regulate a vast array of signalling pathways to control cell proliferation, migration, survival and differentiation. PPIns are differentially present in various sub-cellular compartments and, through the recruitment and regulation of specific proteins, are key regulators of compartment identity and function. Phosphoinositides and the enzymes that synthesise and degrade them are also present in the nuclear membrane and in nuclear membraneless compartments such as nuclear speckles. Here we discuss how PPIns in the nucleus are modulated in response to external cues and how they function to control downstream signalling. Finally we suggest a role for nuclear PPIns in liquid phase separations that are involved in the formation of membraneless compartments within the nucleus.

Summary Phosphoinositides (PI) are important signaling molecules in the nucleus that influence gene expression. However, if and how nuclear PI directly affects the transcriptional machinery is not known. We report that the lipid kinase PIP4K2B regulates nuclear PI5P and the expression of myogenic genes during myoblast differentiation. A targeted screen for PI interactors identified the PHD finger of TAF3, a TATA box binding protein-associated factor with important roles in transcription regulation, pluripotency, and differentiation. We show that the PI interaction site is distinct from the known H3K4me3 binding region of TAF3 and that PI binding modulates association of TAF3 with H3K4me3 in vitro and with chromatin in vivo. Analysis of TAF3 mutants indicates that TAF3 transduces PIP4K2B-mediated alterations in PI into changes in specific gene transcription. Our study reveals TAF3 as a direct target of nuclear PI and further illustrates the importance of basal transcription components as signal transducers., Graphical Abstract, Highlights • PIP4K2B regulates nuclear PI5P and myogenic gene expression during differentiation • A screen identifies 17 of 32 PHD fingers interacting with phosphoinositides • The basal transcription component TAF3 interacts strongly with phosphoinositides • TAF3 transduces changes in nuclear phosphoinositides into transcriptional output, Bultsma et al. show that the basal transcriptional complex protein TAF3 directly binds phosphoinositides and transduces changes in nuclear phosphoinositides into differential transcriptional output that affects myoblast differentiation. The lipid kinase PIP4K2B, phosphoinositides, and TAF3 form a conserved nuclear signaling pathway that selectively regulates transcription.

Lipid signalling in human disease is an important field of investigation and stems from the fact that phosphoinositide signalling has been implicated in the control of nearly all the important cellular pathways including metabolism, cell cycle control, membrane trafficking, apoptosis and neuronal conduction.Adistinct nuclear inositide signalling metabolism has been identified, thus defining a new role for inositides in the nucleus, which are now considered essential co-factors for several nuclear processes, including DNA repair, transcription regulation, and RNA dynamics. Deregulation of phoshoinositide metabolism within the nuclear compartment may contribute to disease progression in several disorders, such as chronic inflammation, cancer, metabolic, and degenerative syndromes. In order to utilize these very druggable pathways for human benefit there is a need to identify how nuclear inositides are regulated specifically within this compartment and what downstream nuclear effectors process and integrate inositide signalling cascades in order to specifically control nuclear function. Here we describe some of the facets of nuclear inositide metabolism with a focus on their relationship to cell cycle control and differentiation.

Previous data suggest the existence of discrete pools of inositol lipids, which are components of a nuclear phosphoinositide (PI) cycle. However, it is not known whether the contents of these pools are regulated during cell proliferation. In the present study we demonstrate that the mass levels of three important constituents of the nuclear PI cycle are regulated during the cell cycle. Radioactive label incorporation into PtdIns(4,5)P(2) was seen to increase dramatically as synchronized cells entered S-phase. This did not coincide with any significant changes in the nuclear mass levels of this lipid, suggesting that the rate of turnover of this molecule was increased. Levels of PtdIns4P, the major substrate for PtdIns(4,5)P(2) production by Type I PtdInsP kinases (PIPkins), were regulated during the cell cycle and indicated a complex relationship between these two lipids. An alternative substrate for PtdIns(4,5)P(2), PtdIns5P, phosphorylated by Type II PIPkins, was present in nuclei at much smaller amounts than the PtdIns4P, and thus is unlikely to contribute significantly to PtdIns(4,5)P(2) turnover. However, a large increase in nuclear PtdIns5P mass was observed when murine erythroleukaemia cells are in G(1), and this could represent a potential pool of nuclear inositol lipid that has a specific signalling role. Analysis of extracted lipid fractions indicated the absence of any PtdIns3P in these nuclei.

Type II phosphatidylinositol phosphate kinases (PIPkins) have recently been found to be primarily phosphatidylinositol 5-phosphate 4-kinases, and their physiological role remains unclear. We have previously shown that a Type II PIPkin [isoform(s) unknown], is localized partly in the nucleus [Divecha, Rhee, Letcher and Irvine (1993) Biochem. J. 289, 617-620], and here we show, by transfection of HeLa cells with green-fluorescent-protein-tagged Type II PIPkins, that this is likely to be the Type IIbeta isoform. Type IIbeta PIPkin has no obvious nuclear localization sequence, and a detailed analysis of the localization of chimaeras and mutants of the alpha (cytosolic) and beta PIPkins shows that the nuclear localization requires the presence of a 17-amino-acid length of alpha-helix (alpha-helix 7) that is specific to the beta isoform, and that this helix must be present in its entirety, with a precise orientation. This resembles the nuclear targeting of the HIV protein Vpr, and Type IIbeta PIPkin is apparently therefore the first example of a eukaryotic protein that uses the same mechanism.

The DNA-dependent protein kinase (DNA-PK) is a nuclear protein serine/threonine kinase that must bind to DNA double-strand breaks to be active. We and others have shown that it is a multiprotein complex comprising an approx. 465 kDa catalytic subunit (DNA-PKcs) and a DNA-binding component, Ku. Notably, cells defective in DNA-PK are hypersensitive to ionizing radiation. Thus X-ray-sensitive hamster xrs-6 cells are mutated in Ku, and rodent V3 cells and cells of the severe combined immune-deficient (Scid) mouse lack a functional DNA-PKcs. Cloning of the DNA-PKcs cDNA revealed that it falls into the phosphatidylinositol (PI) 3-kinase family of proteins. However, biochemical assays indicate that DNA-PK contains no intrinsic lipid kinase activity, but is instead a serine/threonine kinase. We have also found that DNA-PK activity can be inhibited by the PI 3-kinase inhibitors wortmannin and LY294002. Consistent with its proposed role in genome surveillance and the detection of DNA damage, DNA-PKcs is most similar to a subset of proteins involved in cell-cycle checkpoint control and signalling of DNA damage. Furthermore, the recent cloning of the gene mutated in ataxia-telangiectasia (A-T) patients, named ATM (A-T mutated), has revealed that the product of this gene is also a PI 3-kinase family member and is related to DNA-PKcs. Although much is known about the clinical symptoms and cellular phenotypes that arise from disruption of the A-T gene, little is known about the biochemical action of ATM in response to DNA damage. Given its sequence similarity with DNA-PKcs, we speculate that ATM may function in a manner similar to DNA-PK.

DAG, 1,2-diacylglycerol; EGF, epidermal growth factor; TNF, tumour necrosis factor; IGF-I, insulinlike growth factor I; Ins(n)P, inositol phosphate; Ins(n,n)P2, inositol bisphosphate; Ins(n,n,n)P3, inositol trisphosphate; MEL, murine erythroleukemia (Friend); PIC, phosphoinositidase C (phosphatidylinositol- specific phospholipase C); PKC, protein kinase C; PKB, protein kinase B; PLC, phospholipase C; PLD, phospholipase D; PMA, phorbol 12- myristate 13-acetate; PtdCho, phosphatidylcholine; PtdEtn, phosphatidylethanolamine; PtdIns, phosphatidylinositol; PtdIns(x)P, phosphatidylinositol monophosphate, phosphorylated at position x; PtdIns(x,y)P2, phosphatidylinositol bisphosphate, phosphorylated at position x and y; PtdIns(n,n,n)P3, phosphatidylinositol trisphosphate; PtdOH, phosphatidic acid.

Regulatory T cells (Tregs) play fundamental roles in maintaining peripheral tolerance to prevent autoimmunity and limit legitimate immune responses, a feature hijacked in tumor microenvironments in which the recruitment of Tregs often extinguishes immune surveillance through suppression of T-effector cell signaling and tumor cell killing. The pharmacological tuning of Treg activity without impacting on T conventional (Tconv) cell activity would likely be beneficial in the treatment of various human pathologies. PIP4K2A, 2B, and 2C constitute a family of lipid kinases that phosphorylate PtdIns5P to PtdIns(4,5)P2. They are involved in stress signaling, act as synthetic lethal targets in p53-null tumors, and in mice, the loss of PIP4K2C leads to late onset hyperinflammation. Accordingly, a human single nucleotide polymorphism (SNP) near the PIP4K2C gene is linkedwith susceptibility to autoimmune diseases. How PIP4Ks impact on human T cell signaling is not known. Using ex vivo human primary T cells, we found that PIP4K activity is required for Treg cell signaling and immunosuppressive activity. Genetic and pharmacological inhibition of PIP4K in Tregs reduces signaling through the PI3K, mTORC1/S6, and MAPK pathways, impairs cell proliferation, and increases activation-induced cell death while sparing Tconv. PIP4K and PI3K signaling regulate the expression of the Treg master transcriptional activator FOXP3 and the epigenetic signaling protein Ubiquitin-like containing PHD and RING finger domains 1 (UHRF1). Our studies suggest that the pharmacological inhibition of PIP4K can reprogram human Treg identity while leaving Tconv cell signaling and T-helper differentiation to largely intact potentially enhancing overall immunological activity.

Polyphosphoinositides (PPIns) and their modulating enzymes are involved in regulating many important cellular functions including proliferation, differentiation or gene expression, and their deregulation is involved in human diseases such as metabolic syndromes, neurodegenerative disorders and cancer, including Acute Myeloid Leukemia (AML). Given that PPIns regulating enzymes are highly druggable targets, several studies have recently highlighted the potential of targeting them in AML. For instance many inhibitors targeting the PI3K pathway are in various stages of clinical development and more recently other novel enzymes such as PIP4K2A have been implicated as AML targets. PPIns have distinct subcellular organelle profiles, in part driven by the specific localisation of enzymes that metabolise them. In particular, in the nucleus, PPIns are regulated in response to various extracellular and intracellular pathways and interact with specific nuclear proteins to control epigenetic cell state. While AML does not normally manifest with as many mutations as other cancers, it does appear in large part to be a disease of dysregulation of epigenetic signalling and many novel therapeutics are aimed at reprogramming AML cells toward a differentiated cell state or to one that is responsive to alternative successful but limited AML therapies such as ATRA. Here, we propose that by combining bioinformatic analysis with inhibition of PPIns pathways, especially within the nucleus, we might discover new combination therapies aimed at reprogramming transcriptional output to attenuate uncontrolled AML cell growth. Furthermore, we outline how different part of a PPIns signalling unit might be targeted to control selective outputs that might engender more specific and therefore less toxic inhibitory outcomes.

Phosphatidylinositol (PI)-related signaling plays a pivotal role in many cellular aspects, including survival, cell proliferation, differentiation, DNA damage, and trafficking. PI is the core of a network of proteins represented by kinases, phosphatases, and lipases which are able to add, remove or hydrolyze PI, leading to different phosphoinositide products. Among the seven known phosphoinositides, phosphatidylinositol 5 phosphate (PI5P) was the last to be discovered. PI5P presence in cells is very low compared to other PIs. However, much evidence collected throughout the years has described the role of this mono-phosphoinositide in cell cycles, stress response, T-cell activation, and chromatin remodeling. Interestingly, PI5P has been found in different cellular compartments, including the nucleus. Here, we will review the nuclear role of PI5P, describing how it is synthesized and regulated, and how changes in the levels of this rare phosphoinositide can lead to different nuclear outputs.

Polyphosphoinositides (PPIns) are a family of seven lipid messengers that regulate a vast array of signalling pathways to control cell proliferation, migration, survival and differentiation. PPIns are differentially present in various sub-cellular compartments and, through the recruitment and regulation of specific proteins, are key regulators of compartment identity and function. Phosphoinositides and the enzymes that synthesise and degrade them are also present in the nuclear membrane and in nuclear membraneless compartments such as nuclear speckles. Here we discuss how PPIns in the nucleus are modulated in response to external cues and how they function to control downstream signalling. Finally we suggest a role for nuclear PPIns in liquid phase separations that are involved in the formation of membraneless compartments within the nucleus.

The immune system is a complex network that acts to protect vertebrates from foreign microorganisms and carries out immunosurveillance to combat cancer. In order to avoid hyper-activation of the immune system leading to collateral damage tissues and organs and to prevent self-attack, the network has the intrinsic control mechanisms that negatively regulate immune responses. Central to this negative regulation are regulatory T (T-Reg) cells, which through cytokine secretion and cell interaction limit uncontrolled clonal expansion and functions of activated immune cells. Given that positive or negative manipulation of T-Regs activity could be utilised to therapeutically treat host versus graft rejection or cancer respectively, understanding how signaling pathways impact on T-Regs function should reveal potential targets with which to intervene. The phosphatidylinositol-3-kinase (PI3K) pathway controls a vast array of cellular processes and is critical in T cell activation. Here we focus on phosphoinositide 3-kinases (PI3Ks) and their ability to regulate T-Regs cell differentiation and function.

Phosphoinositides are implicated in virtually all aspects of cellular welfare. They help to maintain the structure of biomembranes, but are also essential signal transduction molecules. This is illustrated by PtdIns(4,5)P2, a versatile and important phosphoinositide that regulates many cellular processes including cell survival, calcium homeostasis, vesicular trafficking, cytokinesis, stress responses and cell adhesion. Cells use various enzymes to regulate PtdIns(4,5)P2 levels, including lipid kinases, lipid phosphatases and lipases. PtdIns(4,5)P2 can be synthesized by two closely related families of lipid kinases. Phosphatidylinositol-4-phosphate (PtdIns4P) 5-kinase (PIP5K) phosphorylates PtdIns4P and phosphatidylinositol-5-phosphate (PtdIns5P) 4-kinase (PIP4K) phosphorylates PtdIns5P to make PtdIns(4,5)P2. Cellular PtdIns4P is approximately ten times more abundant than PtdIns5P and therefore PtdIns(4,5)P2 is likely generated by PIP5K mainly. In vivo studies in animals and in mammalian cells suggest that PIP4Ks primarily regulate the level of PtdIns5P, although they may also regulate a specific pool of PtdIns(4,5)P2. PtdIns5P is the latest addition to the ‘palet’ of phosphoinositides and it is the least understood one. In this thesis, I have examined biochemical and functional aspects of PtdIns5P and the PIP4Ks that determine its levels in the cell. I have investigated how PtdIns5P levels change in response to redox signaling, or in response to the activity of the isoforms PIP4K2A and PIP4K2B and I have evaluated PIP4K2B expression in breast tumours.

The control of phosphatidylinositol 3, 4, 5-trisphosphate [PtdIns(3,4,5)P3] level depends on the activities of both PI kinase and PtdIns(3,4,5)P3 phosphatases: 5-phosphatase like SHIP1 and SHIP2, and 3-phosphatase like PTEN. The ubiquitous SH2 domain containing inositol 5-phosphatase SHIP2 contains both a series of protein interacting domains and the ability to dephosphorylate PtdIns(3,4,5)P3. Previous reports obtained in SHIP2 deficient mice have shown that SHIP2 is involved in the control of insulin sensitivity and reducing weight gain on fatty diet. Since SHIP2 is a lipid phosphatase as well as a docking protein, the initial aim that emerged in the lab was to measure the inositol lipid levels in SHIP2 +/+ and deficient cells and compare the levels of 3-phosphoinositides PtdIns(3,4,5)P3 and PtdIns(3,4)P2. At first, we developed mouse embryonic fibroblasts (MEF) as a cellular model. Amongst various stimuli tested, surprisingly, only serum showed an obvious difference in terms of PtdIns(3,4,5)P3 level. This lipid was significantly up regulated in SHIP2 -/- cells but only after short-term (i.e. 5-10 min) incubation with serum. The difference in PtdIns(3,4,5)P3 levels in heterozygous fibroblast cells was intermediate between the +/+ and -/- cells. Serum stimulated PI3K activity appeared to be comparable between +/+ and -/- cells. Moreover, PKB, but not MAP kinase activity, was also potentiated in SHIP2 deficient cells stimulated by serum. The up regulation of PKB activity in serum stimulated cells was totally reversed in the presence of the PI3K inhibitor LY-294002, in both +/+ and -/- cells.Reactive oxygen species (ROS) have emerged as physiological mediators of many cellular responses. H2O2 mimics some effects of insulin in a number of cell culture systems. It also inactivates tyrosine phosphatase activities including PTEN. In addition, in Swiss 3T3 fibroblasts, Gray et al reported that exposure of the cells to H2O2 resulted a huge increase in PtdIns(3,4)P2 level. The authors suspected that the effect was attributed to a inositol 5-phosphatase activity. We thus exposed our cells to H2O2 in order to address the question of the role of SHIP2 in response to oxidative stress.We worked on the same SHIP2 MEF model, stimulated by H2O2: at 15 min, PtdIns(3,4,5)P3 was markedly increased in SHIP2 -/- cells as compared to +/+ cells. In contrast, no significant increase in PtdIns(3,4)P2 could be detected at 15 or 120 min incubation of the cells with H2O2 (0.6 mM). PKB activity was upregulated in SHIP2 -/- cells in response to H2O2 and therefore follows the regulation of PtdIns(3,4,5)P3. As for serum, the PI3K activity appeared to be comparable between +/+ and -/- cells. The levels of PTEN and type I 4-phosphatase [an enzyme that acts on PtdIns(3,4)P2] remained unchanged between the two types of cells. SHIP2 add back experiments in SHIP2 -/- cells confirm its critical role in the control of PtdIns(3,4,5)P3 level in response to H2O2: the decrease in PtdIns(3,4,5)P3, observed in SHIP2 expressing cells, was no longer seen in cells infected with a catalytic mutant of this enzyme., Doctorat en sciences biomédicales, info:eu-repo/semantics/nonPublished

Diacylglycerol kinases (DGKs) phosphorylate the second messenger diacylglycerol (DAG) yielding phosphatidic acid (PA). In this thesis, we investigated which structural domains of DGKtheta are required for DGK activity. Furthermore, we showed that DGKzeta binds to and is activated by the Retinoblastoma tumour suppressor protein (pRB) and the pRB-related proteins p107 and p130, key regulators of the cell-cycle, differentiation and apoptosis. The interaction between pRB and DGKzeta is regulated by protein kinase C. We also found a physiological function of DGKzeta in pRB signalling. Mouse embryonic fibroblasts (MEFs) derived from pRB knock-out mice are unable to arrest in G1 phase of the cell-cycle after gamma-irradiation, whereas wild-type MEFs stop cycling to repair their DNA. DGKzeta overexpression in pRB knock-out MEFs, however, reconstituted a gamma-irradiation-induced cell-cycle arrest in a DGK activity-dependent way. As pRB is known to regulate muscle differentiation, we studied the role of DGKzeta in this process. DGKzeta was found to stimulate muscle differentiation in a DGK activity-dependent fashion and enhanced the expression of early- and late muscle-specific genes. Our results suggest that DGKthata and DGKzeta play a crucial role in determining the correct balance between DAG and PA signalling pathways to regulate physiological functions.